Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T-cells generated ex vivo, is a promising strategy to treat cancer. The T-cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T-cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T-cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T-cells has been shown to be successful in treating melanoma. Novel specificities in T-cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. CARs have successfully allowed T-cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010).
The first generation of CAR-modified T cell showed success in pre-clinical trials and has entered phase I clinical trials. Clinical trials have commenced in ovarian cancer, neuroblastoma and various types of leukemia and lymphoma (clinicaltrials.gov). The clinical trials showed little evidence of anti-tumor activity with insufficient activation, persistence and homing to cancer tissue. Diverse studies have reported partial first-generation CARs in the absence of costimulation leads to anergy and failure of in vivo expansion.
To overcome these limitations, second and the third generation CAR-modification T cells were designed in order to enhance the activation signal, proliferation, production of cytokines and effector function of CAR-modified T cell in preclinical trials. Second-generation CARs were developed to incorporate the intracellular domains of one or more costimulatory molecules such as CD28, OX40, and 4-1BB within the endodomain, and these improved antigen-specific T-cell activation and expansion. Third-generation CARs include a combination of costimulatory endodomains. Both the second and the third generation CAR-modified T cell have entered clinical trials now. The first clinical trial, which has involved T-cells expressing a CAR combining an anti-CD19 binding domain with a 4-1BB costimulatory domain and CD3zeta as an activating signaling domain has led some patients to a complete remission, which has been ongoing 10 months after treatment. The CAR-modified T cells were found to expand 3-logs in these patients, infiltrating and lysing cancer tissue. Interestingly, a fraction of these cells displayed a memory phenotype of T cell for preventive tumor relapses. Although these CAR-modified T cell produced significant therapeutic effect, their activity led to life-threatening tumor lysis 3 weeks after the first infusion of CAR-modified T cell.
Recently adverse events were reported which stress the requirement of special precautions while using second and third generation of CAR-modified T cells. One patient died 5 days after cyclophosphamide chemotherapy followed by infusion of CAR-modified T cells recognizing the antigen ERBB2 (HER-2/neu) (Morgan et al. 2010). The toxicity leads to a clinically significant release of pro-inflammatory cytokines, pulmonary toxicity, multi-organ failure and eventual death of the patient. This and other adverse events highlight the need for caution when employing CAR-modified T cells, as unlike antibodies against tumor-associated antigens, these cells are not cleared from the body within a short amount of time.
There are many on-going researches to develop a safer CAR-based immunotherapy. Several studies reports diverse systems which aim to improve the efficacy and safety of T immunotherapy. T-cell mediated immunity in healthy persons includes multiple sequential steps regulated by a balance between co-stimulatory and inhibitory signals that fine-tune the immunity response. The inhibitory signals referred to as immune checkpoints (such as CTLA-4- or PD-1) are crucial for the maintenance of self-tolerance and also to limit immune-mediated collateral tissue damage (Dolan et al, 2014).
Recently, inhibitory chimeric antigen receptors (iCARs) were designed having as objective to put the brakes on T cell function upon encountering off-target cells. The iCAR is made up of an antigen-specific single-chain variable fragment (scFv) fused to a T cell inhibitory signaling domain. Cells expressing a tumor-associated antigen but not a normal-tissue antigen would induce T cell activation, cytotoxicity and cytokine signaling to kill the on-target cells. In a study (Federov et al. 2013), CTLA-4- or PD-1-based iCARs were shown to selectively limit cytokine secretion, cytotoxicity, and proliferation induced through the endogenous T cell receptor or an activating chimeric receptor. Therefore, to function, the iCAR technology relies on a preliminary selection of 2 antigens: one tumor associated antigen and one normal-tissue antigen. Moreover, the inhibitory effect of PD-1 or CTLA-4 is operating only on off-target cells.
Another system is described in Budde et al. (2013) in which a CD20 Chimeric Antigen Receptor is combined with an inducible caspase 9 (iC9) suicide switch. In the application US 2014/0286987, the latter gene is made functional in the presence of the prodrug AP1903 (tacrolimus) by binding to the mutated FK506-binding protein (FKBP1). A clinical trial is ongoing sponsored by the company Bellicum in which the above capsase technology (CaspaCID™) is engineered into GD2 targeted third generation CAR T cells. Viral transduction transfers DNA from a vector into the target cell and the vector-derived DNA directs expression of chemical induction dimerization (CID) and accessory proteins. In presence of the AP1903 drug, there will be a dimerization of the CID proteins, thus turning on the signal cascade. In the event of a serious of life-threatening toxicity caused by the administered T cells, AP1903 will be infused to trigger rapid destruction and elimination of the CaspaCID™-enabled cells. One important characteristic is that this expression is restricted to the cytoplasm of the cell. Moreover, according to the Bellicum's system, there is no possibility to modulate the activity of the T-cells, since the expression of CaspaCID™ in contact with the drug leads to the death of the T-cells. A similar apoptosis-inducing system based on a multimerizing agent is described in the application WO 2014/152177.
There is a need of a CAR-based immunotherapy technology which is able to inhibit or modulate, by addition of a soluble compound, the activation of chimeric antigen receptor (CAR) immune cells without killing them; which is flexible as the effect of the soluble compound can be either intracellular or extracellular, and which is independent of on/off target cells selection.
The present invention here provides such immunotherapy by which activation can be specifically inhibited/modulated in case of their cytotoxicity (i.e. when needed) by administration of a particular soluble compound.